A cell-free approach to identify binding hotspots in plant immune receptors

Plant immune receptors are often difficult to express heterologously, hindering study of direct interactions between these receptors and their targets with traditional biochemical approaches. The cell-free method ribosome display (RD) enables expression of such recalcitrant proteins by keeping each nascent polypeptide chain tethered to its ribosome, which can enhance protein folding by virtue of its size and solubility. Moreover, in contrast to an in planta readout of receptor activity such as a hypersensitive response that conflates binding and signaling, RD enables direct probing of the interaction between plant immune receptors and their targets. Here, we demonstrate the utility of this approach using tomato recognition of Trichoderma viride ethylene-inducing xylanase (EIX) as a case study. Leveraging the modular nature of the tomato LeEIX2 and LeEIX1 leucine-rich repeat (LRR) receptors, we applied an entropy-informed algorithm to maximize the information content in our receptor segmentation RD experiments to identify segments implicated in EIX binding. Unexpectedly, two distinct EIX-binding hotspots were discovered on LeEIX2 and both hotspots are shared with decoy LeEIX1, suggesting that their contrasting receptor functions are not due to differential modes of ligand binding. Given that most plant immune receptors are thought to engage targets via their LRR sequences, this approach should be of broad utility in rapidly identifying their binding hotspots.

A Yeast-Based Functional Assay to Study Plant N-Degron – N-Recognin Interactions

The N-degron pathway is a branch of the ubiquitin-proteasome system where amino-terminal residues serve as degradation signals. In a synthetic biology approach, we expressed ubiquitin ligase PRT6 and ubiquitin conjugating enzyme 2 (AtUBC2) from Arabidopsis thaliana in a Saccharomyces cerevisiae strain with mutation in its endogenous N-degron pathway. The two enzymes re-constitute part of the plant N-degron pathway and were probed by monitoring the stability of co-expressed GFP-linked plant proteins starting with Arginine N-degrons. The novel assay allows for straightforward analysis, whereas in vitro interaction assays often do not allow detection of the weak binding of N-degron recognizing ubiquitin ligases to their substrates, and in planta testing is usually complex and time-consuming.

Control Of Plant Diseases By An Allium-based Antimicrobial Formulation

The objective of this work was to determine the antimicrobial properties of an allium-based antimicrobial formulation named VEG’LYS (https://phytoauxilium.com/) on the growth of plant pathogenic microorganisms such as fungi, oomycetes, and bacteria. Two anthracnose-related species of the fungal genus Colletotrichum, C. gloeosporioides, and C. fragariae, the oomycete Phytophthora cactorum and the bacterium Xanthomonas fragariae associated with strawberry plants and two fungi Alternaria dauci and Botrytis cinerea, associated with carrot plants were tested in vitro. In in planta experiments, A. dauci and B. cinerea were used.. VEG’LYS inhibited the growth of all plant pathogens tested. We found that both curative and preventive in planta treatments with VEG’LYS inhibited the growth of A. dauci and B. cinerea in carrot. Furthermore, after spraying VEG’LYS on carrot plants the expression of the Pathogenesis-related (PR) 10 gene correlated with the magnitude of infection both in treated and untreated plants. Additionally, it has been shown, that the field application of VEG’LYS on strawberry plants results in a reduction of bacterial and fungal pathogens of strawberry fruits stored in refrigerator. In summary, VEG’LYS is a potential resistance inducer that seems to be suitable for use in both curative and preventive treatments to reduce the diseases and rotting of fruits and vegetables caused by different plant pathogens.

Antagonistic Activity of Bacillus spp. Against Fire Blight Disease In vitro and In planta

Fire blight, affecting more than one hundred and thirty species in the Rosaceae, is probably the most destructive disease affecting pear and apple cultivars in many countries. Currently, there are no effective synthetic compounds with systemic properties. Other major problem is the occurrence and spread of strains of Erwinia amylovora with resistance to streptomycin and copper. Taken into consideration the human and environmental health, the use of biocontrol agents either as an alternative or as a supplement within an integrated fire blight management strategy has attracted worldwide attention. In this study, E. amylovora solution of 107 CFU ml-1 was treated with bio-control agents, Bacillus subtilis str. QST 713, B. amyloliquefaciens str. MBI 600 and their mixture (at solution densities of 106, 107 and 108 CFU ml-1 for each one) on Petri dishes, containing King’s B medium and, compared with positive (streptomycin sulphate) and negative (sterile distilled water) controls. In vivo studies were performed on two-year-old apple cv. Gala seedlings grown in 45-cm-diameter pots containing a sterilized mix of soil–sand–peat under controlled greenhouse conditions (85% relative humidity, 25°C temperature and 16h of day light). The plants were irrigated as needed by drip-irrigation and each pot received a mineral solution (NPK: 20–20–20) at 2 g l-1 twice. When plant shoots reached a length of 30-35 cm, bio-control agents, individually and their mixture, were applied to the plants by a hand-sprayer. Obtaining the data, 108 CFU ml-1 of Bacillus spp. suspension mixture showed strongest in vitro antibacterial effect (26mm) among the tested treatments after positive control streptomycin (28.6mm). Parallel to in vitro findings, the mixture was most effective against the pathogen on cv. Gala (66.03%). Findings show that the use of mixture of beneficial microorganisms with individual antagonistic properties against the pathogen can be an effective strategy as a natural alternative to agrochemicals in the scope of good agriculture practices.

RNAi for Western Corn Rootworm Management: Lessons Learned, Challenges, and Future Directions

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is considered one of the most economically important pests of maize (Zea mays L.) in the United States (U.S.) Corn Belt with costs of management and yield losses exceeding USD ~1–2 billion annually. WCR management has proven challenging given the ability of this insect to evolve resistance to multiple management strategies including synthetic insecticides, cultural practices, and plant-incorporated protectants, generating a constant need to develop new management tools. One of the most recent developments is maize expressing double-stranded hairpin RNA structures targeting housekeeping genes, which triggers an RNA interference (RNAi) response and eventually leads to insect death. Following the first description of in planta RNAi in 2007, traits targeting multiple genes have been explored. In June 2017, the U.S. Environmental Protection Agency approved the first in planta RNAi product against insects for commercial use. This product expresses a dsRNA targeting the WCR snf7 gene in combination with Bt proteins (Cry3Bb1 and Cry34Ab1/Cry35Ab1) to improve trait durability and will be introduced for commercial use in 2022.

Development of PCR-Based Race-Specific Markers for Differentiation of Indian Fusarium oxysporum f. sp. cubense, the Causal Agent of Fusarium Wilt in Banana

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc), is the most lethal soil-borne fungal pathogen infecting bananas. Foc race 1 (R1) and 4 (R4) are the two most predominant races affecting the economically important Cavendish group of bananas in India. A total of seven vegetative compatibility groups (VCGs) from three pathogenic races were isolated during our field survey and were found to be highly virulent towards cv. Grande Naine. According to comparative genome analyses, these Indian Foc VCGs were diverse in genomic organization and effector gene profiles. As a result, false-positive results were obtained with currently available molecular markers. In this context, the study has been initiated to develop PCR-based molecular markers for the unambiguous identification of Indian Foc R1 and R4 isolates. Whole-genome sequences of Foc R1 (GCA_011316005.3), Foc TR4 (GCA_014282265.3), and Foc STR4 (GCA_016802205.1), as well as the reference genomes of Foc (ASM799451v1) and F. oxysporum f. sp. lycopersici (Fol; ASM14995v2), were aligned to identify unique variable regions among the Foc races. Using putative chromosome and predicted gene comparison, race-specific unique Foc virulence genes were identified. The putative lineage-specific identified genes encoding products secreted in xylem (SIX) that may be necessary for disease development in the banana. An in silico analysis was performed and primers were designed from a region where sequences were dissimilar with other races to develop a specific marker for Foc R1, R4, TR4, and STR4. These race-specific markers allowed target amplification in the characterized highly virulent Foc isolates, and did not show any cross-amplification to any other Foc races, VCGs or banana pathogens, Fusarium species, and non-pathogenic Fusarium oxysporum isolates. The study demonstrated that the molecular markers developed for all the three Foc races of India could detect the pathogen in planta and up to 0.025 pg µL−1 DNA levels. Thus, the markers developed in this study are novel and could potentially be useful for the accurate diagnosis and detection of the Indian Foc races which are important for the effective management of the disease.

A Metabolomic Study of Epichloë Endophytes for Screening Antifungal Metabolites

Epichloë endophytes, fungal endosymbionts of Pooidae grasses, are commonly utilized in forage and turf industries because they produce beneficial metabolites that enhance resistance against environmental stressors such as insect feeding and disease caused by phytopathogen infection. In pastoral agriculture, phytopathogenic diseases impact both pasture quality and animal production. Recently, bioactive endophyte strains have been reported to secrete compounds that significantly inhibit the growth of phytopathogenic fungi in vitro. A screen of previously described Epichloë-produced antifeedant and toxic alkaloids determined that the antifungal bioactivity observed is not due to the production of these known metabolites, and so there is a need for methods to identify new bioactive metabolites. The process described here is applicable more generally for the identification of antifungals in new endophytes. This study aims to characterize the fungicidal potential of novel, ‘animal friendly’ Epichloë endophyte strains NEA12 and NEA23 that exhibit strong antifungal activity using an in vitro assay. Bioassay-guided fractionation, followed by metabolite analysis, identified 61 metabolites that, either singly or in combination, are responsible for the observed bioactivity. Analysis of the perennial ryegrass-endophyte symbiota confirmed that NEA12 and NEA23 produce the prospective antifungal metabolites in symbiotic association and thus are candidates for compounds that promote disease resistance in planta. The “known unknown” suite of antifungal metabolites identified in this study are potential biomarkers for the selection of strains that enhance pasture and turf production through better disease control.

MicroRNAs Are Involved in Regulating Plant Development and Stress Response through Fine-Tuning of TIR1/AFB-Dependent Auxin Signaling

Auxin, primarily indole-3-acetic acid (IAA), is a versatile signal molecule that regulates many aspects of plant growth, development, and stress response. Recently, microRNAs (miRNAs), a type of short non-coding RNA, have emerged as master regulators of the auxin response pathways by affecting auxin homeostasis and perception in plants. The combination of these miRNAs and the autoregulation of the auxin signaling pathways, as well as the interaction with other hormones, creates a regulatory network that controls the level of auxin perception and signal transduction to maintain signaling homeostasis. In this review, we will detail the miRNAs involved in auxin signaling to illustrate its in planta complex regulation.

A Laminarin-Based Formulation Protects Wheat Against Zymoseptoria tritici Via Direct Antifungal Activity and Elicitation of Host Defense-Related Genes

The present study aimed to evaluate the potential of the laminarin-based formulation Vacciplant® to protect and induce resistance in wheat against Zymoseptoria tritici, a major pathogen on this crop. Under greenhouse conditions, a single foliar spraying of the product two days before inoculation with Z. tritici reduced disease severity and pycnidium density by 42% and 45%, respectively. Vacciplant® exhibited a direct antifungal activity on Z. tritici conidial germination both in vitro and in planta. Moreover, it reduced in planta substomatal colonization as well as pycnidium formation on treated leaves. Molecular investigations revealed that Vacciplant® elicits but did not prime the expression of several wheat genes related to defense pathways, including phenylpropanoids (phenylalanine ammonia-lyase and chalcone synthase), octadecanoids (lipoxygenase and allene oxide synthase), and pathogenesis‐related proteins (β‐1,3‐endoglucanase and chitinase). By contrast, it did not modulate the expression of oxalate oxidase gene involved in the reactive oxygen species metabolism. UHPLC-MS analysis indicated limited changes in leaf metabolome after product application in both non-inoculated and inoculated conditions, suggesting a low metabolic cost associated with induction of plant resistance. This study provides evidence that the laminarin-based formulation confers protection to wheat against Z. tritici through direct antifungal activity and elicitation of plant defense-associated genes.